Abstract In the mammalian cochlea, inner hair cells (IHCs) operate as auditory receptors, while outer hair cells (OHCs) provide the cellular basis for cochlear amplification. It is well established that hair cell mechanotransduction (MET) properties, such as resting open probability, differ significantly between the two cochlear hair cell types. The molecular basis of these differences, and their significance for the respective functions of OHCs and IHCs, are not known. In preliminary studies, we discovered that two isoforms of Myosin VIIa (MYO7A) are expressed in the cochlea, generated by alternative start sites. Analysis of isoform-specific KO mice demonstrated that the longer MYO7A isoform is preferentially expressed in IHCs, while the shorter isoform, which lacks an 11-amino acid N-terminal extension, is predominantly expressed in OHCs. In mice lacking the long isoform, IHCs develop normally but progressively lose the transducing rows of stereocilia (rows 2 and 3). Additionally, these mice suffer profound hearing loss by 9 weeks of age. Remarkably, distortion product otoacoustic emissions (DPOAEs), a measure of OHC function, are unaffected, consistent with an IHC-specific phenotype. The molecular, biochemical and mouse tools we generated provide a strong platform for addressing fundamental questions regarding the function of MYO7A in hair cell MET, particularly in specifying the characteristic differences in MET properties in the two cochlear hair cell types. In Specific Aim 1, we test whether MYO7A is required for hair cell MET. Most models designate MYO7A as a molecular motor integral to the upper tip link density, possibly involved in establishing the mechanical tension important for MET function. However, direct evidence is lacking because knockout and knockdown mouse models of MYO7A have severe defects in hair bundle development. Our long isoform-specific KO mice allow the investigation of MYO7A function in normally developed hair cells. In Specific Aim 2, we will define the enzymatic activities and subcellular localization of MYO7A isoforms in IHCs and OHCs, and then correlate these outcomes with cell type-specific physiological and functional characteristics. Finally, in SA3, we propose to identify cis-and trans-regulatory factors that mediate the cell-type specific expression of MYO7A isoforms. In summary, a novel feature of MYO7A, a well- characterized deafness gene, will enable our exploration of the molecular differences between inner and outer hair cells, an issue of considerable significance for hearing research.